CAPILLARY ZONE ELECTROPHORESIS FOR DETERMINATION OF REDUCED AND OXIDISED ASCORBATE AND GLUTATHIONE IN ROOTS AND LEAF SEGMENTS OF Zea mays PLANTS EXPOSED TO Cd AND Cu
Sławomir Dresler
Maria Curie-Skłodowska UniversityWaldemar Maksymiec
Maria Curie-Skłodowska UniversityAbstract
The concentration of non-enzymatic antioxidants such as ascorbate and glutathione in tissues is one of the major plant responses to biotic and many abiotic stresses, including metals. Therefore, it is crucial to develop the most effective methods for simultaneous quantitative analysis of these antioxidants. Capillary zone electrophoresis allows relatively fast and effective analysis. The aim of the paper was to apply and optimise the capillary electrophoresis conditions for simultaneous determination of glutathione, glutathione disulphide, ascorbate, and dehydroascorbate in small plant tissue samples exposed to copper and cadmium. The method ensures good linearity and reproducibility, with correlation coefficients 0.988 for ascorbate and 0.999 for glutathione and glutathione disulphide, and with detection limits approximately 2.50, 0.65 and 0.50 ppm, respectively. Cu stress was found to increase the ascorbate concentration and glutathione content in leaves, while Cd increased glutathione in the oldest leaf segments and root.
Keywords:
non-enzymatic antioxidants, heavy metal, stress, plantReferences
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Žunić G., Spasić S., 2008. Capillary electrophoresis method opitmized with a factorial design for the determination of glutathione and acid status using human capillary blood. J. Chromatogr. B. 873, 70–76.
Carru C., Zinellu A., Sotgia S., Marongiu G., Farina M.G., Usai M.F., Pes G.M., Tadolini B., Deiana L., 2003. Optimization of the principal parameters for the ultrarapid electrophoretic separation of reduced and oxidized glutathione by capillary electrophoresis. J. Chromatogr. A. 1017, 233–238.
Chao Y.Y., Kao Ch.H., 2010. Heat shock-induced ascorbic acid accumulation in leaves increases cadmium tolerance of rice (Oriza sativa L.) seedlings. Plant Soil. 336, 39–48.
Córdoba-Pedregosa M., Córdoba F., Villalba J.M., González-Reyes J.A., 2003. Zonal changes in ascorbate and hydrogen peroxide contents, peroxidase, and ascorbate-related enzyme activities in onion roots. Plant. Physiol. 131, 679–706.
Davey M.W., Bauw G., Van Montagu M., 1996. Analysis of ascorbate in plant tissues by highperformance capillary zone electrophoresis. Anal Biochem. 239, 8–19.
Davey M.W., Bauw G., Van Montagu M., 1997. Simultaneous high-performance capillary electrophoresis analysis of the reduced and oxidised forms of ascorbate and glutathione. J Chromatogr. B. 697, 269–276.
Deutsch J.C., 2000. Dehydroascorbic acid. J. Chromatogr A. 881, 299–307.
Drążkiewicz M., Skórzyńska-Polit E., Krupa Z., 2003a. Response of the ascorbate-glutathione cycle to excess copper in Arabidopsis thaliana (L.). Plant Sci. 164, 195–202.
Drążkiewicz M., Tukendorf A., Baszyński T., 2003b. Age-dependent response of maize leaf segments to cadmium treatment: Effect on chlorophyll fluorescence and phytochelatin accumulation. J. Plant Physiol. 160, 247–254.
Frazier R.A., Ames J.M., Nursten H.E., 2000. Experimental variables in capillary electrophoresis. In capillary electrophoresis for food analysis, method development. The Royal Society of Chemistry, Cambridge, 16–24.
Galiana-Balaguer L., Roselló S., Herrero-Martínez J.M., Maquieira A., Nuez F., 2001. Determination of L-ascorbic acid in Lycopersicon fruits by capillary zone electrophoresis. Anal. Biochem. 296, 218–224.
Gökmen V., Kahraman N., Demir N., Acar J., 2000. Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in fruit and vegetables. J. Chromatogr A. 881, 309–316.
Havel K., Pritts K., Wielgos T., 1999. Quantitation of oxidized and reduced glutathione in plasma by micellar electrokinetic capillary electrophoresis. J. Chromatogr. A. 853, 215–223.
Herrero-Martínez J.M., Simó-Alfonso E., Deltoro V.I., Calatayud A., Ramis-Ramos G., 1998. Determination of L-ascorbi acid and total ascorbic acid in vascular and nonvascular plants by capillary zone electrophoresis. Anal. Biochem. 265, 275–281.
Herrero-Martínez J.M., Simó-Alfonso E.F., Ramis-Ramos G., 2000. Simultaneous determination of L-ascorbic acid, glutathione, and their oxidized forms in ozone-exposed vascular plants by capillary zone electrophoresis. Environ. Sci. Technol. 34, 1331–1336.
Kacem B., Marshall M.R., Matthews R.F., Gregory J.F., 1986. Simultaneous analysis of ascorbic and dehydroascorbic acid by high-performance liquid chromatography with postcolumn derivatization and UV absorbance. J. Agric. Food. Chem. 34, 271–274.
Krupa Z., Moniak M. 1998. The stage of leaf maturity implicates the response of the photosynthetic apparatus to cadmium toxicity. Plant Sci. 138, 149–156.
Maksymiec W., Baszyński T., 1996. Different susceptibility of runner bean plants to excess copper as a function of the growth stages of primary leaves. J. Plant Physiol. 149, 217–221
Mendoza J., Garrido T., Riveros R., Parada J., 2009. Rapid capillary electrophoresis analysis of glutathione and glutathione disulphide in roots and shoots of plants exposed to copper. Phytochem. Anal. 20, 114–119.
Monostori P., Wittmann G., Karg E., Túri S., 2009. Determination of glutathione and glutathione disulfide in biological samples: an in-depth review. J. Chromatogr. B. 877, 3331–3346
Muscari C., Pappagallo M., Ferrari D., Giordano E., Capanni C., Caldarera C.M., Guarnieri C., 1998. Simultaneous detection of reduced and oxidized glutathione in tissues and mitochondria by capillary electrophoresis. J. Chromatogr. B. 707, 301–307.
Nováková L., Solich P., Solichová D., 2008. HPLC methods for simultaneous determination of ascorbic and dehydroascorbic acids. Trends. Anal. Chem. 27, 942–958
Rellan-Alvarez R., Ortega-Villasante C., Alvarez-Fernandez A., del Campo F.F., Hernadez L.E., 2006. Stress responses of Zea mays to cadmium and mercury. Plant Soil. 279, 41–50.
Tie S.G., Tang Z.J., Zhao Y.M., Li W., 2012. Oxidative damage and antioxidant response caused by excess copper in leaves of maize. Afr. J. Biot. 11, 4378–4384.
Wu F.B., Chen F., Wei K., Zhang G.P., 2004. Effect of cadmium on free amino acid, glutathione and ascorbic acid concentrations in two barley genotypes (Hordeum vulgare L.) differing in cadmium tolerance. Chemosphere 57, 447–454.
Yang Q., Krautmacher C., Schilling D., Pittelkow M.R., Naylor S., 2002. Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis. Biomed. Chromatogr. 16, 224–228.
Zinellu A., Carru C., Sotgia S., Deiana L., 2004. Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection. Anal. Biochem. 330, 298–305.
Zinellu A., Sotgia S., Caddeo S., Deiana L., Carru C., 2005. Sodium glycylglycine as effective electrolyte run buffer for ascorbic and uric acid separation by CZE: A comparison with two other CE assays. J. Separ. Sci., 28, 16, 2193–2199.
Žunić G., Spasić S., 2008. Capillary electrophoresis method opitmized with a factorial design for the determination of glutathione and acid status using human capillary blood. J. Chromatogr. B. 873, 70–76.
Sławomir Dresler
Maria Curie-Skłodowska University
Maria Curie-Skłodowska University
Waldemar Maksymiec
Maria Curie-Skłodowska University
Maria Curie-Skłodowska University
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